Chromosome 5 of Human Pathogen Candida albicans Carries Multiple Genes for Negative Control of Caspofungin and Anidulafungin Susceptibility.

crossmark Chromosome 5 of Human Pathogen Candida albicans Carries Multiple Genes for Negative Control of Caspofungin and Anidulafungin Susceptibility Sumanun Suwunnakorn, Hironao Wakabayashi, Elena Rustchenko Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, USA Candida albicans is an important fungal pathogen with a diploid genome that can adapt to caspofungin, a major drug from the echinocandin class, by a reversible loss of one copy of chromosome 5 (Ch5). Here, we explore a hypothesis that more than one gene for negative regulation of echinocandin tolerance is carried on Ch5. We constructed C. albicans strains that each lacked one of the following Ch5 genes: CHT2 for chitinase, PGA4 for glucanosyltransferase, and CSU51, a putative transcription factor. We demonstrate that independent deletion of each of these genes increased tolerance for caspofungin and anidulafungin, another echinocandin. Our data indicate that Ch5 carries multiple genes for negative control of echinocandin tolerance, although the final number has yet to be established. C andida albicans is a unicellular budding fungus that lives as part of normal human gut or genital microflora. It is also a major opportunistic pathogen in immunocompromised individuals. Naturally occurring strains of C. albicans are usually diploids with eight pairs of chromosomes. However, aneuploidy is well tolerated and is a means to introduce phenotypic diversity in a cell population (1). Moreover, the copy number of a particular chromosome can control adaptation to a specific adverse environment (1), including the development of resistance to fluconazole, a major antifungal from the azole class (1–3). The best-studied regulation due to chromosome copy number is the reversible loss of chromosome 5 (Ch5) controlling resistance to L-sorbose, a toxic sugar that kills C. albicans or other fungi in a manner similar to that of echinocandins (reviewed in reference 4). This regulation is complex, including multiple CSU (control of sorbose utilization) genes scattered along Ch5 that are organized in two functionally redundant pathways (5). The expression of at least two such genes, CSU51 (orf19.1105.2) and CSU53 (orf19.3931), is finely tuned by antisense regulation (6). Recently, we used laboratory mutants to demonstrate that the reversible loss of Ch5 also controls tolerance to the major echinocandin caspofungin, such that strains with one copy acquire caspofungin tolerance whereas strains that spontaneously duplicate monosomic Ch5 revert to susceptibility (4). Based on the model system of sorbose resistance, Ch5 can carry multiple genes encoding negative regulators of echinocandin susceptibility. Unlike research into the negative control of sorbose resistance, the study of negative control of echinocandin tolerance due to loss of one Ch5 is in its beginning. It was previously reported that disruption of both copies of the Ch5 gene PGA4 (orf19.4035) confers increased caspofungin tolerance (7). PGA4 encodes a glycosylphosphatidylinositol (GPI)-anchored cell surface protein called 1,3-␤-D-glucanosyltransferase, which resembles the GEL family of oligosaccharide transferases in Aspergillus fumigatus. However, this result needs reevaluation as the gene was disrupted in the genetic background of the BWP17 strain, which is unstable and responds to genetic manipulations in a nonconventional fashion (8). Most importantly, one Ch5 in BWP17 lacks an ⬃36.8-kb portion adjacent to the right telomere (8) that encompasses PGA4. December 2016 Volume 60 Number 12 Another Ch5 gene that is a strong candidate for negative control is CHT2 (orf19.3895), which encodes a GPI-anchored chitinase involved in hydrolysis of cell wall chitin. CHT2 is repressed in the core caspofungin response (9, 10). Of a total of four C. albicans genes for chitinases, only CHT2 and CHT3 (orf19.7586) were reported to be downregulated after treatment of C. albicans biofilm with micafungin, another echinocandin, which allowed the authors to suggest that CHT2 and CHT3 are involved in the cell wall’s tolerance to stress caused by micafungin and the induction of chitin synthesis (11). Earlier, mutations of CHT2 and CHT3 were found in a laboratory mutant that became highly tolerant to caspofungin and exhibited high chitin content but had no FKS1 mutations causing clinical caspofungin resistance (12). The authors suggested that mutations of CHT2 and CHT3 could result in increased chitin and could affect susceptibility to caspofungin. In this work, we prepared and characterized deletion strains lacking an entire open reading frame (ORF) of either PGA4 or CHT2 and deletion strains lacking another putative GPI anchor, CSU51. The latter encodes a predicted transcription factor of the helix-loop-helix class, which, as described above, was previously found to be a negative regulator of sorbose resistance (5, 6). We demonstrated that independent deletion of PGA4, CHT2, or CSU51 conferred increased tolerance to the echinocandins caspofungin and anidulafungin. Our data indicate that C. albicans Ch5 carries multiple genes for the negative control of susceptibility to Received 29 August 2016 Returned for modification 23 September 2016 Accepted 4 October 2016 Accepted manuscript posted online 10 October 2016 Citation Suwunnakorn S, Wakabayashi H, Rustchenko E. 2016. Chromosome 5 of human pathogen Candida albicans carries multiple genes for negative control of caspofungin and anidulafungin susceptibility. Antimicrob Agents Chemother 60:7457–7467. doi:10.1128/AAC.01888-16. Address correspondence to Elena Rustchenko, . Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01888-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved. Antimicrobial Agents and Chemotherapy aac.asm.org 7457 Suwunnakorn et al. TABLE 1 C. albicans strains used in this study Strain Genotype Source CAF4-2 ER503 (fragmentation site 12) ER506 (fragmentation site 12) NCS8 (csu51⫹/⫺) NCS6 (csu51⫹/⫺) NCS5 (csu51⫹/⫺) NACS1 (csu51⫺/⫺) NACS8 (csu51⫺/⫺) NACS19 (csu51⫺/⫺) NC136 (cht2⫹/⫺) NC72 (cht2⫹/⫺) NC133 (cht2⫹/⫺) NAC4 (cht2⫺/⫺) NAC12 (cht2⫺/⫺) NAC7 (cht2⫺/⫺) NP6 (pga4⫹/⫺) NP3 (pga4⫹/⫺) NP5 (pga4⫹/⫺) NAP88 (pga4⫺/⫺) NAP86 (pga4⫺/⫺) NAP76 (pga4⫺/⫺) JRCT1 JMC200-3-3 ura3⌬::imm434/ura3⌬::imm434 394.22-kb truncation of right arm of Ch5 Same as above, but independent truncation csu51⌬::URA3-FLPa/CSU51 csu51⌬::URA3-FLP/CSU51 csu51⌬::URA3-FLP/CSU51 csu51⌬::URA3-FLP/csu51⌬::NAT1-FLPb csu51⌬::URA3-FLP/csu51⌬::NAT1-FLP csu51⌬::URA3-FLP/csu51⌬::NAT1-FLP cht2⌬::URA3-FLP/CHT2 cht2⌬::URA3-FLP/CHT2 cht2⌬::URA3-FLP/CHT2 cht2⌬::URA3-FLP/cht2⌬::NAT1-FLP cht2⌬::URA3-FLP/cht2⌬::NAT1-FLP cht2⌬::URA3-FLP/cht2⌬::NAT1-FLP pga4⌬::URA3-FLP/PGA4 pga4⌬::URA3-FLP/PGA4 pga4⌬::URA3-FLP/PGA4 pga4⌬::URA3-FLP/pga4⌬::NAT1-FLP pga4⌬::URA3-FLP/pga4⌬::NAT1-FLP pga4⌬::URA3-FLP/pga4⌬::NAT1-FLP Clinical isolate Same as above, but a single Ch5, MTL␣ (...truncated)